CN102885011A - Novel method for infecting silkworms with recombinant baculovirus - Google Patents
Novel method for infecting silkworms with recombinant baculovirus Download PDFInfo
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- CN102885011A CN102885011A CN2012104203928A CN201210420392A CN102885011A CN 102885011 A CN102885011 A CN 102885011A CN 2012104203928 A CN2012104203928 A CN 2012104203928A CN 201210420392 A CN201210420392 A CN 201210420392A CN 102885011 A CN102885011 A CN 102885011A
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Abstract
The invention discloses a novel method for infecting silkworms with recombinant baculovirus. The method comprises the following steps: silkworm larvae are gathered into a cultivation frame; in a standard that each 100 silkworm larvae are ejected with 5ml of recombinant baculovirus liquid, the recombinant baculovirus liquid with 106pfu is ejected to the body surfaces of the silkworm larvas; and the silkworm larvae are not fed with mulberry leaves, and after most part of the baculovirus liquid is dried, the silkworm larvae are fed in an ordinary method till the silkworm larvae have the symptom of virus infection. The method disclosed by the invention has the advantages that a traditional inoculation method of percutaneous infection which is used directly is changed, the inoculation operation is simplified, the inoculation time is saved, and the working efficiency of an insect baculovirus expression system is improved.
Description
Technical field
The present invention relates to the recombinant virus infection technology, relate in particular to the method for a kind of recombinant virus infection silkworm.
Background technology
Insect bculovrirus expression vector system (BacuLovirus Expression Vector System, be called for short BEVS) is to utilize the recombinant baculovirus that carries the external source genes of interest to make carrier in insect bodies or insect cell culture carries out a recombinant protein production system of Expression product.Owing to having, this system can utilize individual or its cultured cell of insect to carry out the advantage such as large-scale efficient Expression product to become at present one of the most effective eukaryotic expression system.At present BEVS mainly comprise American-European countries just the autographa california nuclear polyhedrosis virus of extensive use (
Autographa californicaNucleopolyhedrosis virus is called for short AcNPV) and China, day this application more bombyx mori nuclear polyhydrosis virus (
Bombyx moriNucleopolyhedrosis virus is called for short BmNPV) two class expression systems (Smith et al., 1983; Maeda et al., 1984,1985).The silkworm BmNPV expression system since the economic insects-silkworm that can utilize China's number of animals raised maximum as expression vector, thereby have advantages of that cost is low, be fit to large-scale production, have very much China's characteristic (Lv Hongsheng, 1998).Yet, present inoculation technique all is to adopt the method that recombinant virus is injected directly in the Silkworm, Bombyx mori to infect expression, in actual mechanical process owing to needing one by one silkworm to inject, spended time is long, complex operation, and owing to silkworm in injection process owing to reasons such as polypide wrigglings, cause virus liquid to flow out external, finally cause infecting unsuccessfully, therefore at present traditional percutaneous injection method defective is obvious, and operating efficiency is low.In order to improve the operating efficiency of baculovirus expression system, be necessary to develop more efficiently mode of infection.By repetition test, we find virus liquid is applied to the silkworm larva body surface, also can realize the cell entry polypide, finally cause Viral infection, reach with percutaneous injection and infect the same effect.
Summary of the invention
The object of the present invention is to provide a kind of recombinant baculovirus through the new method of skin infected silkworm.
In order to achieve the above object, the technical solution adopted in the present invention is as follows:
Recombinant Bombyx mori baculovirus of the present invention through the method for skin infected silkworm is: silkworm larva is concentrated on raise in the frame; Spray 5ml recombinant Bombyx mori baculovirus liquid as standard take 100 boss's silkworm larvas, will contain 10
6The recombinant Bombyx mori baculovirus liquid of pfu sprays the body surface in silkworm larva; Spray the mulberry leaf of not feeding in rear 1 hour, after most of virus liquid drying, raise silkworm larva according to conventional method, until the virus infections symptom appears in silkworm larva.
Further, silkworm larva of the present invention is 3 ages or 4 ages.
Compared with prior art, the beneficial effect that has of the present invention is:
(1) the inventive method result of implementation shows, by recombinant Bombyx mori baculovirus liquid being sprayed the body surface in silkworm larva, also can make in the cell entry Silkworm, Bombyx mori; Because silkworm is the same with other insects, and an open blood circulation system is arranged, in case cell entry blood, virus just can infect very soon, and towards periphery tissue diffusion, finally realizes general infection.
(2) silkworm-baculovirus expression system might become a powerful in China's biotechnology industry, the present invention has solved the problem of recombinant virus infection complex operation, inefficiency preferably, simplify the virus infections method, greatly improved the operating efficiency of baculovirus expression system.
Description of drawings
(1) Fig. 1 is the photo that the blood sample of silkworm larva is observed under the fluorescence inverted microscope, and wherein, Fig. 1 (a) is the blood sample of FFI silkworm larva; Fig. 1 (b) is the blood sample of the metainfective silkworm larva of reorganized silkworm baculovirus, and green fluorescence occurs.
(2) Fig. 2 is the symptom contrast figure without the silkworm larva that infects the infection of silkworm larva and reorganized silkworm baculovirus, and wherein, Fig. 2 (a) is without infecting silkworm larva; Fig. 2 (b) is the silkworm larva that reorganized silkworm baculovirus infects.
Embodiment
Below be described in further detail the inventive method:
1. material: recombinant Bombyx mori baculovirus adopts at present very ripe technique construction, concrete grammar can be referring to the paper of delivering both at home and abroad, as being published in " utilizing bacterial transposon to make up the Bac-to-Bac rapid gene expression system that is applicable to a silkworm " literary composition of " silkworm industry science " in 2006,32(2): 183-188.Utilize recombinant Bombyx mori baculovirus of expressing reporter gene green fluorescent protein EGFP of Bac-to-Bac technique construction of above-mentioned silkworm, utilize this virus, use the inventive method through the skin infected silkworm larva.
In following examples, use silkworm hybrid, variety name is QiuFeng×BaiYu, and silkworm larva is raised with mulberry leaf, and raising temperature is 23～25 ℃.
In following examples, recombinant Bombyx mori baculovirus is as follows through the method for skin infected silkworm:
3 ages of silkworm or 4 instar larvaes are concentrated on raise in the frame; Spray the 5ml virus liquid as standard take 100 boss silkworms, will contain 10 with miniaturised nebuliser
6Pfu(virus infections unit) recombinant Bombyx mori baculovirus liquid sprays the body surface in silkworm larva, and at the injection mulberry leaf of not feeding in rear 1 hour; After most of or whole viral liquid dried, raise silkworm according to conventional method, until the virus infections symptom shown in Fig. 2 (b) appears in silkworm larva afterwards.
Infectious effect is measured: after body surface 72-96 hour that recombinant Bombyx mori baculovirus liquid is sprayed in silkworm larva, a small amount of blood of getting silkworm larva places on the slide, under the fluorescence inverted microscope, observe and the green fluorescence [shown in Fig. 1 (b)] that virus infections produces whether occurs, if have, illustrate that then the reorganized silkworm baculovirus of this silkworm larva infects.Spray in about 120 hours behind the body surface of silkworm larva at recombinant Bombyx mori baculovirus liquid, further the disease symptom of observer's silkworm larva.
Interpretation of result: recombinant Bombyx mori baculovirus liquid is being sprayed in about 96 hours behind the body surface of silkworm larva, when under the fluorescence inverted microscope, observing the blood of observer's silkworm larva, can find obvious green fluorescence [referring to Fig. 1 (b)]; The blood sample of the silkworm larva of uninfecting virus then can not produce fluorescence [referring to Fig. 1 (a)].
Spray in about 120 hours behind the body surface of silkworm larva at recombinant Bombyx mori baculovirus liquid, can find that typical virus infections symptom appears in silkworm larva, such as anorexia, body surface is shinny, body segment swelling, manic creep etc. [contrast Fig. 2 (a) and Fig. 2 (b)] further specifies silkworm larva by virus infections, and recombinant protein-green fluorescent protein has obtained expression.
Claims (2)
1. a recombinant Bombyx mori baculovirus is characterized in that: silkworm larva is concentrated on raise in the frame through the method for skin infected silkworm; Spray 5ml recombinant Bombyx mori baculovirus liquid as standard take 100 boss's silkworm larvas, will contain 10
6The recombinant Bombyx mori baculovirus liquid of pfu sprays the body surface in silkworm larva; Spray the mulberry leaf of not feeding in rear 1 hour, after most of virus liquid drying, raise silkworm larva according to conventional method, until the virus infections symptom appears in silkworm larva.
2. recombinant Bombyx mori baculovirus according to claim 1 is characterized in that through the method for skin infected silkworm: described silkworm larva is 3 ages or 4 ages.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1229135A (en) * | 1999-04-02 | 1999-09-22 | 中国农业科学院蚕业研究所 | Method for producing phytase using silkworm biological reactor |
| CN1410540A (en) * | 2001-09-29 | 2003-04-16 | 中国农业科学院蚕业研究所 | Method of producing broad adaptability, high specific activity phytase and acidic phosphoesterase using silkworm bioreactor |
| CN1456665A (en) * | 2002-05-10 | 2003-11-19 | 安徽固源生物工程有限责任公司 | Production of organic phosphorous chemicals decomposing enzyme by silkworm bioreactor |
| US20050074432A1 (en) * | 2003-10-03 | 2005-04-07 | Tzong-Yuan Wu | Insect larva aerosol infection method for producing recombinant proteins and baculovirus bio-insecticides |
| CN1696294A (en) * | 2004-05-11 | 2005-11-16 | 中国农业科学院生物技术研究所 | High-performance bioreactor for baculovirus of insects |
| CN1974776A (en) * | 2006-12-07 | 2007-06-06 | 浙江大学 | Method of infecting silkworm with recombinant baculovirus |
| CN101280021A (en) * | 2008-05-20 | 2008-10-08 | 广西天池海洋生物药业有限公司 | Engineered protein against shrimp white spot syndrome virus, preparation and use thereof |
| CN101307317A (en) * | 2008-06-25 | 2008-11-19 | 中国农业科学院兰州兽医研究所 | Method for preparing rabies virus antigen |
| CN101396024A (en) * | 2008-11-04 | 2009-04-01 | 武汉武大绿洲生物技术有限公司 | Dendrolimus punctatus cytoplasmic polyhedrosis virus(dpcpv) bacillus thuringiensis wettable powder and preparation method thereof |
| CN102286533A (en) * | 2010-06-18 | 2011-12-21 | 财团法人工业技术研究院 | Insect infection method for production of proteins |
| CN102613250A (en) * | 2012-03-01 | 2012-08-01 | 武汉武大绿洲生物技术有限公司 | Preparation method of pieris rapae granulosis virus active compound |
-
2012
- 2012-10-29 CN CN2012104203928A patent/CN102885011A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1229135A (en) * | 1999-04-02 | 1999-09-22 | 中国农业科学院蚕业研究所 | Method for producing phytase using silkworm biological reactor |
| CN1410540A (en) * | 2001-09-29 | 2003-04-16 | 中国农业科学院蚕业研究所 | Method of producing broad adaptability, high specific activity phytase and acidic phosphoesterase using silkworm bioreactor |
| CN1456665A (en) * | 2002-05-10 | 2003-11-19 | 安徽固源生物工程有限责任公司 | Production of organic phosphorous chemicals decomposing enzyme by silkworm bioreactor |
| US20050074432A1 (en) * | 2003-10-03 | 2005-04-07 | Tzong-Yuan Wu | Insect larva aerosol infection method for producing recombinant proteins and baculovirus bio-insecticides |
| CN1696294A (en) * | 2004-05-11 | 2005-11-16 | 中国农业科学院生物技术研究所 | High-performance bioreactor for baculovirus of insects |
| CN1974776A (en) * | 2006-12-07 | 2007-06-06 | 浙江大学 | Method of infecting silkworm with recombinant baculovirus |
| CN101280021A (en) * | 2008-05-20 | 2008-10-08 | 广西天池海洋生物药业有限公司 | Engineered protein against shrimp white spot syndrome virus, preparation and use thereof |
| CN101307317A (en) * | 2008-06-25 | 2008-11-19 | 中国农业科学院兰州兽医研究所 | Method for preparing rabies virus antigen |
| CN101396024A (en) * | 2008-11-04 | 2009-04-01 | 武汉武大绿洲生物技术有限公司 | Dendrolimus punctatus cytoplasmic polyhedrosis virus(dpcpv) bacillus thuringiensis wettable powder and preparation method thereof |
| CN102286533A (en) * | 2010-06-18 | 2011-12-21 | 财团法人工业技术研究院 | Insect infection method for production of proteins |
| CN102613250A (en) * | 2012-03-01 | 2012-08-01 | 武汉武大绿洲生物技术有限公司 | Preparation method of pieris rapae granulosis virus active compound |
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Application publication date: 20130123 |